The present invention is directed to xcex2-polypeptide molecules which are oligomers and polymers of xcex2-amino acids having stable and well-defined secondary structures, including helices and sheets, methods of generating combinatorial libraries using these xcex2-polypeptides, and combinatorial libraries formed thereby.
Chemists have long sought to extrapolate the power of biological catalysis and recognition to synthetic systems. These efforts have focused largely on low molecular weight catalysts and receptors. Most biological systems, however, rely almost exclusively on large polymers such as proteins and RNA to perform complex chemical functions.
Proteins and RNA are unique in their ability to adopt compact, well-ordered conformations. These two biopolymers are unique also because they can perform complex chemical operations (e.g., catalysis, highly selective recognition, etc.). Folding is linked to function in both proteins and RNA because the creation of an xe2x80x9cactive sitexe2x80x9d requires proper positioning of reactive groups. Consequently, there has been a long-felt need to identify synthetic polymer backbones which display discrete and predictable folding propensities (hereinafter referred to as xe2x80x9cfoldamersxe2x80x9d) to mimic natural biological systems. Such backbones will provide molecular xe2x80x9ctoolsxe2x80x9d to probe the functionality of large-molecule interactions (e.g. protein-protein and protein-RNA interactions).
Much work on xcex2-amino acids and peptides synthesized therefrom has been performed by a group led by Dieter Seebach in Zurich, Switzerland. See, for example, Seebach et al. (1996) Helv. Chim. Acta. 79:913-941: and Seebach et al. (1996) Helv. Chim. Acta. 79:2043-2066. In the first of these two papers Seebach et al. describe the synthesis and characterization of a xcex2-hexapeptide, namely (H-xcex2-HVal-xcex2-HAla-xcex2-HLeu)2-OH. Interestingly, this paper specifically notes that prior art reports on the structure of xcex2-peptides have been contradictory and xe2x80x9cpartially controversial.xe2x80x9d In the second paper, Seebach et al. explore the secondary structure of the above-noted xcex2-hexapeptide and the effects of residue variation on the secondary structure.
Dado and Gellman (1994) J. Am. Chem. Soc. 116:1054-1062 describe intramolecular hydrogen bonding in derivatives of xcex2-alanine and xcex3-amino butyric acid. This paper postulates that xcex2-peptides will fold in manners similar to xcex1-amino acid polymers if intramolecular hydrogen bonding between nearest neighbor amide groups on the polymer backbone is not favored.
Suhara et al. (1996) Tetrahedron Lett. 37(10):1575-1578 report a polysaccharide analog of a xcex2-peptide in which D-glycocylamine derivatives are linked to each other via a C-1 xcex2-carboxylate and a C-2 xcex1-amino group. This class of compounds has been given the trivial name xe2x80x9ccarbopeptoids.xe2x80x9d
Regarding methods to generate combinatorial libraries, several recent reviews are available. See, for instance, Ellman (1996) Acc. Chem. Res. 29:132-143 and Lam et al. (1997) Chem. Rev. 97:411-448.
The present invention is drawn to a genus of conformationally-restricted xcex2-polyamides which strongly favor a helical or sheet secondary structure and which can serve as building blocks for stable tertiary structures. These stable secondary structures include helices analogous to the well-known xcex1-helical structure seen in xcex1-amino acids. Several of the subject compounds assume helical secondary structures stabilized by hydrogen bonding every 12th or 14th atom of the backbone (12-helix and 14-helix, respectively). Still other compounds according to the invention contain a xe2x80x9creverse turnxe2x80x9d residue which mimics the reverse turn often seen in anti-parallel sheet structure seen in conventional peptides and proteins. These xcex2-peptides according to the invention exhibit an anti-parallel secondary sheet structure.
More specifically, the invention is directed to xcex2-polypeptides comprising compounds of formula: 
wherein Z is a single bond or a substituent selected from the group consisting of: 
and wherein when Z is a single bond, X and Y combined, together with the carbon atoms to which they are bonded, define a substituted or unsubstituted C3-C8 cycloalkyl, cycloalkenyl or heterocyclic ring having one or more nitrogen atoms as the sole heteroatom, the substituents being selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl; hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl, amino, mono- or di-C1-C6-alkylamino, carboxamido, carboxamido-C1-C6-alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, heteroaryl-C1-C6-alkyl, and combinations thereof; and
when Z is not a single bond, X and Y combined, together with the carbon atoms to which they are bonded, are as defined above or X and Y are independently selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl; hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl, amino, mono- or di-C1-C6alkylamino, carboxamido, carboxamido-C1-C6alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, heteroaryl-C1-C6alkyl, and combinations thereof; and m, n, and p are positive integers.
The invention is also directed to an array comprising a plurality of polypeptides, the polypeptides comprising compounds of formula: 
wherein Z is a single bond or a substituent selected from the group consisting of: 
and wherein when Z is a single bond, X and Y combined, together with the carbon atoms to which they are bonded, define a substituted or unsubstituted C3-C8 cycloalkyl, cycloalkenyl or heterocyclic ring having one or more nitrogen atoms as the sole heteroatom, the substituents being selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl; hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl, amino, mono- or di-C1-C6-alkylamino, carboxamido, carboxamido-C1-C6-alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, and heteroaryl-C1-C6-alkyl; and
when Z is not a single bond, X and Y combined, together with the carbon atoms to which they are bonded, are as defined above or X and Y are independently selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl; hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl, amino, mono- or di-C1-C6-alkylamino, carboxamido, carboxamido-C1-C6-alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, and heteroaryl-C1-C6-alkyl; and
m, n, and p are positive integers;
the peptides at selected, known locations on a substrate or in discrete solutions, wherein each of the polypeptides is substantially pure within each of the selected known locations or discrete solutions and has a composition which is different from other polypeptides disposed at other selected and known locations on the substrate or in other discrete solutions.
The invention is also directed to a method for preparing a combinatorial library of xcex2-polypeptides, the method comprising at least two successive iterations of:
(a) covalently linking a first subunit via its C terminus to a plurality of separable solid substrates, the first subunit selected from the group consisting of 
wherein X and Y combined, together with the carbon atoms to which they are bonded, define a substituted or unsubstituted C3-C8 cycloalkyl, cycloalkenyl or heterocyclic ring having one or more nitrogen atoms as the sole heteroatom, the substituents being selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl; hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl, amino, mono- or di-C1-C6-alkylamino, carboxamido, carboxamido-C1-C6-alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, and heteroaryl-C1-C6-alkyl;
R1 and R2 are independently selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl; hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl, amino, mono- or di-C1-C6-alkylamino, carboxamido, carboxamido-C1-C6-alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, and heteroaryl-C1-C6-alkyl; and
Pg is a protecting group; then
(b) randomly dividing the plurality of substrates into at least two sub-groups; and
(c) deprotecting the first subunits attached to the at least two sub-groups; then
(d) in separate and independent reactions, covalently linking to the first subunit of each of the at least two sub-groups a second subunit independently selected from the group listed in step (a), provided that at least one of the first or second subunits is selected from the group consising of 
xe2x80x83wherein X, Y, and Pg are as defined above; then
(e) combining the at least two sub-groups into a single plurality; and then
(f) repeating steps (b) through (e).
The invention is also directed to xe2x80x9cmixedxe2x80x9d xcex2-polypeptides comprising compounds of formula 
xe2x80x83wherein X and Y combined, together with the carbon atoms to which they are bonded, n, and p are as defined above, and R1 and R2 are independently selected from the group consisting of hydroxy, linear or branched C1-C6-alkyl, alkenyl, alkynyl: hydroxy-C1-C6-alkyl, amino-C1-C6-alkyl, C1-C6-alkyloxy, C1-C6-alkyloxy-C1-C6-alkyl. amino, mono- or di-C1-C6-alkylamino, carboxamido, carboxamido-C1-C6-alkyl, sulfonamido, sulfonamido-C1-C6-alkyl, urea, cyano, fluoro, thio, C1-C6-alkylthio, mono- or bicyclic aryl, mono- or bicyclic heteraryl having up to 5 heteroatoms selected from N, O, and S; mono- or bicyclic aryl-C1-C6-alkyl, heteroaryl-C1-C6-alkyl, and combinations thereof; and
The invention is further directed to a method for preparing a combinatorial library of xcex2-polypeptides, the method comprising at least two successive iterations of first covalently linking a first subunit via its C terminus to a plurality of separable solid substrates, the first subunit selected from the group consisting of 
wherein X and Y combined, together with the carbon atoms to which they are bonded, R1, and R2 are as defined above, and Pg is a protecting group. And then randomly dividing the plurality of substrates into at least two sub-groups and deprotecting the first subunits attached to the at least two sub-groups. Then in separate and independent reactions, covalently linking to the first subunit of each of the at least two sub-groups a second subunit independently selected from the above-listed group, provided that at least one of the first or second subunits is selected from the group consising of 
wherein X, Y, and Pg are as defined above. Then combining the at least two sub-groups into a single plurality.
The invention is further drawn to a combinatorial library of xcex2-polypeptides comprising a plurality of different xcex2-polypeptides, each xcex2-polypeptide covalently linked to a solid support, the combinatorial library produced by the process described immediately above.
Another embodiment of the invention is drawn to an array comprising a plurality of xcex2-polypeptides as described above at selected, known locations on a substrate or in discrete solutions, wherein each of the polypeptides is substantially pure within each of the selected known locations and has a composition which is different from other polypeptides disposed at other selected and known locations on the substrate.
The primary advantage of the present invention is that it allows the construction of synthetic peptides of known secondary structures having high conformational stability. These synthetic polyamides have utility in investigating the biological interactions involving biopolymers. The predictable and stable secondary structure of the present compounds allows them to mimic natural protein secondary structure, thereby allowing targeted disruption of large-molecule interactions (e.g., protein-protein interactions.)
The switch in helical hydrogen bond directionality between the xcex2-peptide 12- and 14-helices (See FIGS. 2A and 2B) is unprecedented among xcex1-peptides. The residue-based conformational control offered by xcex2-peptides, which was predicted computationally, makes this class of unnatural foldamers well suited for molecular design efforts, e.g., generation of novel tertiary structures, and combinatorial searches for selective biopolymer ligands.
As a natural consequence, the invention is further drawn to the use of these synthetic polyamides as base molecules from which to synthesize large libraries of novel compounds utilizing the techniques of combinatorial chemistry. In addition to varying the primary sequence of the xcex2-amino acid residues, the ring positions of these compounds (and notably the equitorial positions in the cyclohexyl-rigidified xcex2-amino acids) can be substituted with a wide variety of substituents, including hydroxy, linear or branched C1-C6-alkyl, C1-C6-alkyloxy, amino, mono- or di-C1-C6-alkylamino, carboxamido, sulfonamido, urea, cyano, fluoro, thio, C1-C6-alkylthio, and the like. The main advantage here is that substituents placed on the backbone rings do not substantially alter the secondary structure of the peptide. Consequently, the subject compounds can be utilized to contruct vast libraries having different substituents, but all of which share a stabilized secondary structure in both the solid state and in solution.
Other aims, objects, and advantages of the invention will appear more fully from a complete reading of the following Detailed Description of the Invention and the attached claims.